Isolation and purification of nucleic acids play a central role in modern molecular biology, and increasingly in medicine. Both laboratory and diagnostic research require the use of nucleic acids in gene cloning and genetic analysis. Many of these techniques require keeping ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) pure and free of contamination. In many instances, the availability of small amounts of starting sample material poses a problem during isolation of the nucleic acid. The limited amount of sample material makes the need to limit loss of the sample material a critical concern.
A known method for isolating nucleic acids from a small amount of starting material includes the use of a spin filter column (“filter column”) that contains a nucleic acid binding material (i.e., a filter). Examples of binding material/filters include silicas like glass powder, silica particles, glass microfibers, diatomaceous earth, etc. These filters are often associated with a “filter surface area.” This filter surface area is not limited to the surface area of a side of the filter. Instead, since the filters are usually comprised of microscopic fibers, particles, porous substances, etc., the filter surface area is actually defined by the surface area of the components, which comprise the filter. For example, a filter that comprises glass microfibers may have a filter surface area defined by the surface area of the microfibers within the filter (either all of the microfibers or a portion thereof).
In some cases, filter columns may isolate nucleic acids directly from cells or biological tissue. In the first step a filter column is inserted into a microcentrifuge tube (e.g., a 1.5 ml tube) and a solution containing nucleic acids along with undesirable impurities is loaded into the top of the filter column. Depending upon the application, the starting material containing the nucleic acids is prepared from cells that have been treated with a disrupting solution causing the release of the nucleic acids. Alternatively, the nucleic acid solution is the product of an earlier reaction step. In either case, the nucleic acid binds to the filter column filter in the presence of a chaotropic agent. Then the filter column is centrifuged in a microcentrifuge. Centrifugation forces the solution through the filter column's filter and binds the nucleic acid to the filter. Next, the filter with the nucleic acids bound therein is washed by applying a washing solution to the top of the filter column and centrifuging again. After each wash the filter column can be removed from the collection tube so that the material in the collection tube may be removed. Finally, placement of an elution buffer (usually water having a specific pH) at the top of the column and applying centrifugation elutes the nucleic acid that is bound to the filter. Given the proper pH, the nucleic acid dissolves and elutes with the liquid into the collection tube. It is important to note that the volumes of the binding and wash solutions can be relatively large, thus necessitating the use of a larger (1.5-2.0 mL) tube. On the other hand, the volumes for elution are often smaller, making it desirable to have a smaller tube for the elution step. Eluting directly into a smaller tube allows one to proceed to the next reaction step in the small tube, rather than having to pipette out of the large tube. It is desirable to use a small tube in downstream processes. As discussed herein, use of a pipette is undesirable as it introduces the risk of loss of samples as well as contamination of the sample. Moreover, as described herein, there are additional benefits in keeping the sample solution in a smaller tube.
Several companies provide kits that include filter columns designed to use this technique for isolating nucleic acids. QIAGEN, Promega, and Boehringer Mannheim GmbH offer filter columns based on the above-described principle. However, existing filter columns cannot be used interchangeably with collection tubes of different sizes. Instead, these previously known filter columns only fit into a single size collection tube (e.g., a standard 1.5-2.0 mL microcentrifuge tube.)
This limitation presents a problem as many applications may benefit if a single filter column could be interchanged with collection tubes of multiple sizes. For example, given a small amount of nucleic acid in the starting material, it is best to elute the purified nucleic acid into a very small volume of fluid so that nucleic acid does not become too dilute in the fluid. Obviously, the resulting combination of elution buffer and nucleic acid will occupy a small volume. Many applications that require processing of nucleic acid may benefit when storing this small volume of material in a smaller sized collection tube. For instance, an application such as amplification of the purified DNA by polymerase chain reaction (PCR) requires placement of the nucleic acid into a thin-walled 0.5 mL or 0.2 mL microcentrifuge tube.
Accordingly, it may seem ideal to use a smaller filter column, which is specifically designed to fit a 0.5 mL or 0.2 mL microcentrifuge tube. However, a significant drawback is that these smaller filter columns limit the amount of wash and binding solution that can be passed through the column with each bind/wash. This limitation necessitates additional wash steps and increased handling of the filter column and microcentrifuge tube, thus presenting an undesirable increased risk of contamination. Also, as discussed above, the requirement of large volumes of binding and wash solutions often necessitate the use of a larger collection tubes (e.g., a 1.5-2.0 mL microcentrifuge tube.)
The remaining alternative is to use a filter column specifically designed to fit into a larger collection tube (e.g., a 1.5 mL-2.0 mL microcentrifuge tube.) While this alternative minimizes the additional wash steps and increased handling discussed above, the alternative presents additional problems. For instance, after purification, the nucleic acid solution must be eluted from the filter column into an appropriately sized 1.5 mL-2.0 mL microcentrifuge tube. As discussed above, many applications may benefit by storing the elution buffer and nucleic acid in a smaller sized microcentrifuge tube. Consequently, the elution buffer and nucleic acid must then be transferred (e.g., by aspiration) into a smaller (e.g. 0.5 mL) tube. Again, this extra transfer step introduces the undesirable potentials of contamination and loss of some of the nucleic acid.
The present invention involves the elution of purified nucleic acids into small volumes (generally less than 20 microliters). The filters of the filtration columns described are adapted to optimally function with small elution volumes by minimizing fluidic holdup during the purification and elution procedures, and by tailoring the filter characteristics.
In view of the above, there remains a need to use different-sized collection tubes with a single filter column. The ability to use different-sized collection tubes with a single filter column, particularly when dealing with small elution volumes, overcomes the problems associated with the existing art.
The invention described herein addresses the problems discussed above. Moreover, the invention described herein allows centrifugation from one filter column into at least two distinct sizes of collection tubes. The disclosed invention may be used with commercially available collection tubes.